This invention pertains to derivatized biotin compounds, and to methods of using those compounds.
Biotin (also known as vitamin H) is an essential growth factor found in all animals, plants, fungi, and bacteria. It is found, for example, bound to proteins or polypeptides in the liver, pancreas, kidney, milk, and in yeasts. Biotin is a cofactor for a group of enzymes that catalyze carboxylation reactions, transcarboxylation reactions, and decarboxylation reactions. The reactions catalyzed by biotin-dependent enzymes are involved in several essential metabolic pathways, including gluconeogenesis, fatty acid synthesis, and amino acid catabolism. All biotin-dependent carboxylases catalyze their respective reactions via the two step reaction shown below:
Enzyme-biotin+Mg2++ATP+HCO3xe2x88x92⇄Enzyme-biotin-CO2xe2x88x92+Mg2++ADP+Pixe2x80x83xe2x80x83(1)
Enzyme-biotin-CO2xe2x88x92+acceptor⇄acceptor-CO2xe2x88x92+Enzyme-biotinxe2x80x83xe2x80x83(2)
R. Guchhait et al., J. Biol. Chem. vol. 249, pp. 6646-6656 (1974) showed that in the first partial reaction shown above, biotin is carboxylated on the 1xe2x80x2-N. Since bicarbonate is the source of CO2 for all biotin-dependent carboxylases, carboxylation of the 1xe2x80x2-N of biotin is accomplished by activating bicarbonate through phosphorylation with ATP to form a carboxyphosphate intermediate. The carboxyl group is then transferred from the carboxyphosphate intermediate to biotin to form carboxybiotin.
In the field of biotechnology, biotin has become an important reagent in methods to label, detect, and purify proteins and nucleic acids. These methods are based on the remarkable affinity between biotin and the proteins avidin and streptavidin. The dissociation constant of biotin from avidin or streptavidin is about 10xe2x88x9215M, one of the strongest known interactions between a protein and a ligand. While all parts of the biotin molecule contribute to this tight binding, hydrogen bonding donation by the ureido nitrogens is the major contributor.
Q. Han et al., xe2x80x9cSynthesis of (+)-Biotin Derivatives as HIV-1 Protease Inhibitors,xe2x80x9d Bioorg. and Med. Chem., vol. 6, pp. 1371-1374 (1996) reported the synthesis of several bis-N-alkylated biotin derivatives, and their activity against HIV-1 protease. All biotin derivatives reported in this paper were substituted on both urea nitrogens. There was no suggestion to selectively substitute biotin at one of the nitrogen atoms only, nor was there any suggestion of how such a selective synthesis might be performed.
Up to 25% diacylated biotin products have previously been reported following acylation of biotin methyl ester with (1) methylchloroformate or (2) trifluoroacetic anhydride. See (1) J. Knappe et al., Biochemishe Zeitschrift, vol. 335, pp. 168-176 (1961); and (2) A. Berkessel et al., Bioorganic Chem., vol. 14, pp. 249-261 (1986); respectively.
P. Tipton et al., xe2x80x9cCatalytic Mechanism of Biotin Carboxylase: Steady-State Kinetic Investigations,xe2x80x9d Biochemistry, vol. 27, pp. 4317-4325 (1988) reported that no biotin analog that had been tested had inhibited biotin carboxylase (at page 4322).
The ureido ring of biotin is of great importance in binding to avidin, and the 1xe2x80x2-N of that ring is directly involved in biotin""s role as a carboxytransfer intermediate. However, there have been relatively few studies involving functionalization of the 1xe2x80x2-N of biotin. Most prior research on derivatizing biotin has involved acylation chemistry at the carboxylic acid terminus of the pendant alkyl chain.
We have discovered a method to functionalize biotin at the 1xe2x80x2-N, selectively and with high efficiency. The resulting 1xe2x80x2-N-substituted biotin has an electrophilic xe2x80x9chandlexe2x80x9d that is amenable to reaction with a wide variety of nucleophiles to generate a new family of biotin analogs. Such nucleophiles might include, for example, acetyl coenzyme A, benzyl alcohol, benzoic acid, aniline, or other nucleophiles known in the art.
This synthesis is more selective for functionalization at the 1xe2x80x2-N of biotin than have been any known prior syntheses. Compounds prepared from the 1xe2x80x2-N-substituted biotin are useful in inhibiting various enzymatic reactions, including the inhibition of HIV protease.
As initial examples, we have synthesized compounds 1 and 3 (see FIGS. 1 and 2). Compound 1 is formally derived from phosphonoacetic acid coupled to the 1xe2x80x2-N of biotin. The synthesis of Compound 3 was highly efficient and selective: a 98% yield with essentially 100% selectivity, in a synthesis conducted on the multi-gram scale. The synthesis of Compound 1 had an overall yield of 35%, and a 100% selectivity.
We have shown that compound 1 acts as a stable analog of the carboxyphosphate intermediate in naturally-occurring biotin-mediated CO2 transfer. Compound 1 inhibits the activity of the biotin carboxylase component of the enzyme acetyl CoA carboxylase. This is the first reported biotin-derived inhibitor of biotin carboxylase.
The synthesis of Compound 3 may readily be scaled up to perform large-scale, selective acylations of biotin to form Compound 1 or other end products.